CN114846023A - Maltopentaose/maltohexaose variant alpha-amylases - Google Patents

Abstract

The present invention discloses compositions and methods relating to maltopentaose/maltohexaose alpha-amylases. Variant alpha-amylases can be used, for example, in starch liquefaction and saccharification, for cleaning starch stains in laundry, dishwashing, and other applications, in textile processing (eg, desizing), in animal feed for improving digestibility, As well as for baking and brewing.

Description

maltopentaose/maltohexaose variant alpha-amylase

technical field

The present invention discloses compositions and methods involving maltopentaose/maltohexaose variant alpha-amylases. Variant alpha-amylases are useful, for example, in cleaning starch stains, starch liquefaction and saccharification, textile desizing, baking, and brewing.

Background technique

Starch consists of a mixture of amylose (15%-30% w/w) and amylopectin (70%-85% w/w). Amylose is composed of straight chains with alpha-1,4-linked glucose units having a molecular weight (MW) of from about 60,000 to about 800,000. Amylopectin is a branched polymer containing alpha-1,6 branch points per 24-30 glucose units; its MW can be as high as 100 million.

Alpha-amylases hydrolyze starch, glycogen and related polysaccharides by randomly cleaving internal alpha-1,4-glycosidic bonds. Alpha-amylases, especially from Bacilli, have been used for a variety of different purposes, including starch liquefaction and saccharification, textile desizing, starch modification in the paper and pulp industry, brewing, baking , production of syrups for the food industry, production of raw materials for fermentation processes, and in animal feed to increase digestibility. These enzymes can also be used to remove starchy soils and stains during dishwashing and laundry.

The products produced by alpha-amylase hydrolysis of starch vary in the number of consecutive glucose molecules. Most commercial alpha-amylases produce a range of products from glucose (G1) to maltoheptaose (G7). For reasons that are not entirely clear, alpha-amylases that produce large amounts of maltopentaose and maltohexaose appear to be particularly useful for certain commercial applications, including incorporation into detergent cleaning compositions. Numerous publications have described mutations in maltopentaose/maltohexaose producing alpha-amylases and others. Nonetheless, there remains a need for more robust and better-performing engineered alpha-amylase molecules.

SUMMARY OF THE INVENTION

The compositions and methods of the present invention relate to maltopentaose/maltohexaose variant amylase polypeptides and methods of use thereof. Aspects and examples of the compositions and methods of the present invention are summarized in the following separately numbered paragraphs:

1. In one aspect, there is provided a recombinant, non-naturally occurring parent variant alpha-amylase molecule comprising a mutation at position 91 numbered with reference to SEQ ID NO: 1 and the bottom of the alpha-amylase TIM barrel structure Mutations at the amino acid residues at the bottom defined as residues 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322 , 323, 324, 325, 362, 363, and 364, wherein the wild-type amino acid residue present at position 28 of the parent molecule is positively charged.

2. In some embodiments of the variant alpha-amylase of paragraph 1, the mutation at position 91 is a substitution of a naturally occurring residue for a positively charged residue.

3. In some embodiments of the variant alpha-amylase of paragraph 1 or 2, the mutation at position 91 is a substitution of a naturally occurring residue for an arginino group (ie, X91R).

4. In some embodiments of the variant alpha-amylase of any of paragraphs 1-3, the at least one mutation at the bottom of the alpha-amylase TIM barrel structure is selected from the group consisting of: X40N, X40D, X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L, and X364M.

5. In some embodiments of the variant alpha-amylase of any of paragraphs 1-4, the at least one mutation at the bottom of the alpha-amylase TIM barrel structure is selected from the group consisting of: T40N, T40D, Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L, and Y364M.

6. On the other hand, provide a kind of recombinant, non-naturally occurring variant alpha-amylase, it comprises the arginine at position 91 and is not present in the following characteristics in the naturally occurring alpha-amylase. At least one of: N or D at position 40, F or L at position 100, Y at position 263, D, K, or Q at position 288, R, N, or M at position 324, or L or M.

7. In some embodiments, the variant alpha-amylase of any of paragraphs 1-6 further comprises a mutation at a residue in a loop comprising a surface numbered with reference to SEQ ID NO: 1 Residues 167, 169, 171, 172, and 176 exposed.

8. In some embodiments of the variant alpha-amylase of paragraph 7, at least one mutation in the loop is selected from the group consisting of X167F, X169H, X171Y, X172R, X172N, and X176S.

9. In some embodiments of the variant alpha-amylase of paragraph 8, at least one mutation in the loop is selected from the group consisting of: W167F, Q169H, R171Y, Q172R, Q172N, and R176S.

10. In some embodiments, the variant alpha-amylase of any one of paragraphs 1-6 further comprises F at position 167, H at position 169, position numbered with reference to SEQ ID NO: 1 Y at 171, R or N at position 172, or S at position 176.

11. In another aspect, there is provided a recombinant, non-naturally occurring variant alpha-amylase comprising a mutation at position 172 and a mutation at position 288 numbered with reference to SEQ ID NO:1.

12. In another aspect, there is provided a recombinant, non-naturally occurring variant alpha-amylase comprising an arginine or asparagine at position 172 and an asparagine at position 288 numbered with reference to SEQ ID NO:1. Aspartic acid.

13. In some embodiments, the variant alpha-amylase of any of paragraphs 1-12 further comprises mutations at positions 116 and/or 281 numbered with reference to SEQ ID NO:1.

14. In some embodiments, the variant alpha-amylase of any of paragraphs 1-12 further comprises an arginine at position 116 or a serine at position 281 numbered with reference to SEQ ID NO: 1 .

15. In some embodiments, the variant alpha-amylase of any of paragraphs 1-14 further comprises mutations at positions 190 and/or 244 numbered with reference to SEQ ID NO:1.

16. In some embodiments, the variant alpha-amylase of any of paragraphs 1-14 has a proline at position 190 and/or a proline at position 244 numbered with reference to SEQ ID NO: 1. amino acid, glutamic acid or glutamine.

17. In some embodiments, the variant alpha-amylase of any of paragraphs 1-16 further comprises at least two of R181, G182, T183, and G184 using SEQ ID NO: 1 deletion of residues.

18. In some embodiments, the variant alpha-amylase of any of paragraphs 1-16 further comprises a pairwise deletion of residues corresponding to R181 and G182 or residues T183 and G184.

19. In another aspect, there is provided a recombinant, non-naturally occurring variant alpha-amylase comprising:

Substitutions of (i) numbered using SEQ ID NO: 1 are selected from the group consisting of:

(a) X40N-X91R-X169H-X183M-X281N,

(b) X172R-X190P-X288D,

(c) X172R-X244E-X288D-X474R,

(d) X91R-X172R-X190P-X324M,

(e) X40N-X91R-X190P-X263Y,

(f) X40N-X91R-X244E-X364L,

(g) X91R-X172R-X190P-X324R,

(h) X91R-X116R-X172R-X244E-X281S-X288D,

(i) X40N-X91R-X100F-X116R-X172N-X244Q-X281S,

(j) X40N-X91R-X172R-X244Q-X263Y-X281S,

(k) X91R-X172R-X190P-X324N,

(l) X40D-X91R-X172R-X190P-X281S-X324R, and

(m)X364L; and

(ii) pairwise deletion of residues selected from the group consisting of residues corresponding to:

181 and 182, and

183 and 184.

20. In some embodiments, the variant alpha-amylase of paragraph 19 comprises:

Substitutions of (i) numbered using SEQ ID NO: 1 are selected from the group consisting of:

(a) T40N-S91R-Q169H-T183M-H281N,

(b) Q172R-E190P-S288D,

(c) Q172R-S244E-S288D-S474R,

(d) S91R-Q172R-E190P-I324M,

(e) T40N-S91R-E190P-F263Y,

(f) T40N-S91R-S244E-Y364L,

(g) S91R-Q172R-E190P-I324R,

(h) S91R-W116R-Q172R-S244E-H281S-S288D,

(i) T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S,

(j) T40N-S91R-Q172R-S244Q-F263Y-H281S,

(k)S91R-Q172R-E190P-I324N,

(l) T40D-S91R-Q172R-E190P-H281S-I324R, and

(m) Y364L; and

(ii) pairwise deletion of residues selected from the group consisting of:

R181 and G182, and

T183 and G184.

21. On the other hand, there is provided a recombinant, non-naturally occurring variant alpha-amylase comprising three or more of the following features: (a) D or N at position 40 and/or position 91 R at position 100, and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and/or L at position 364, optionally with ( c) H at position 169, M at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R, and/or a combination of R at position 116, (d) optionally in combination with (e) paired deletions at positions 181 and 182 or 183 and 184, in all cases using SEQ ID NO: 1 number.

22. In some embodiments, the variant alpha-amylase of any of paragraphs 1-21 is combined with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 has at least 70%, at least 80%, at least 90%, or at least 95% amino acid sequence identity.

23. In another aspect, there is provided a detergent composition comprising the variant alpha-amylase of any of paragraphs 1-22.

24. In some embodiments, the detergent composition of paragraph 23 further comprises a variant subtilisin from Bacillus gibsonii having amino acid substitutions X39E, X99R, X126A, X127E, and X128G, and further comprising one or more additional substitutions numbered with reference to SEQ ID NO:5 selected from the group consisting of: N74D-M211L-N253P, R179Q-M211L-N253P, N74D-N253P, N85R-G160Q-R179Q-M211L-N212S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N212S-N253P, R179Q-M211L, G160Q-R179Q-M211L-N253P, G162-M25179Q-N24 M211L-N242D, G160Q-R179Q-M211L-N212S, N74D-R179Q-M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N74D-Q200L-M211L, N74D-G160Q-N212S M211L-N253P, G160Q-R179Q, G160Q-R179Q-N212S, N74D-G160Q-N253P, N74D-G160Q-R179Q-M211L-N212S-N253P, N74D-N085R-G160Q-R179Q-M21-2-2-G11-G-N74L N253P, N74D-N085R-N116R-Q200L-Q256E, N74D-G160Q-R179Q-N212S-N253P, N74D-G160Q-M211L-N212S, N74D-G160Q, N74D-G160Q-R179Q-M211L, N74D-N253RP N74D-G160Q-N212S, N74D-G160Q-M211L, N74D-G160Q-R179Q-N253P, N74D, N74D-G160Q-R179Q-M211L-N212S, N74D-N085R-M211L-N212S, N74D-G160Q-R17 G160Q-R179Q-M211L, N74D-M211L-Q256E, N74D-G160Q-R179Q, R179Q-M211L-N21 2S-N253P, R179Q-M211L-N212S, N74D-N085R-R179Q-M211L-N212S, N74D-M211L-N212S, N74D-R179Q-M211L-N212S, N74D-M211L-N242D, N74D-Q256E, 74D-Q256E, 74D-Q256E, 74D-Q256E Q200L-M211L-N242D-Q256E, N74D-Q200L, N74D-M211N-N212Q, N74D-M211N-N212Q-Q256E, N74D-M211N-Q256E, N74D-M211Q, N74D-M211Q-N21Q-562Q, N74D-M21 N74D-M211Q-Q256E, N74D-N198A-M211Q, N74D-N198A-M211Q-N212Q, N74D-N198A-M211Q-Q256E, N74D-N198G-M211Q, N74D-N198G-M211Q-N218G-562Q, N74D-2N198G-562Q N74D-N198K-M211Q-N212Q, N74D-N198L-M211Q-N212Q, N74D-N198Q-M211Q-N212Q, N74D-N198R-M211Q-N212Q, N74D-N198T-M211Q-N212Q, N74Q-N1198V-D N212Q-Q256E, N74D-Q256E, N74D-R207Q, N74D-R207Q-M211N, N74D-R207Q-M211N-N212Q, N74D-R207Q-M211N-N212Q-Q256E, N74D-R207Q-M211N-Q256E, N74D-R207Q-M211N-Q256E N74D-R207Q-M211Q-N212Q, N74D-R207Q-M211Q-N212Q-Q256E, N74D-R207Q-N212Q, N74D-R207Q-N212Q-Q256E, N74D-R207Q-11Q256E, N74D-N198S-M207Q-N212Q, and N74D-R207Q-1Q256E , and has at least 90% amino acid sequence identity with SEQ ID NO:6.

25. In another aspect, there is provided a method for converting starch into oligosaccharides, the method comprising making the starch with an effective amount of the variant α- as described in any of paragraphs 1-22. Amylase exposure.

26. In another aspect, there is provided a method for removing starch stains or soils from a surface, the method comprising mixing the surface with an effective amount of variant α as described in any of paragraphs 1-22 - contacting with amylase and allowing the polypeptide to hydrolyze starch components present in the starch stain to produce smaller starch-derived molecules that dissolve in the aqueous composition, thereby removing the starch stain from the surface .

27. In another aspect, a nucleic acid encoding the variant alpha-amylase of any of paragraphs 1-22 is provided.

28. In another aspect, a host cell comprising the nucleic acid of paragraph 27 is provided.

These and other aspects and embodiments of the compositions and methods of the present invention will be apparent from the following specification and accompanying examples.

Description of drawings

Figure 1 shows the Clustal W amino acid sequence alignment of AA2560, AA707, AA560, and AAI10.

Figure 2 shows a view of AA2560 amylase passing through the central β-barrel. Residues at the bottom of the barrel are shown as spherical α-carbon positions. Amino acid numbers for these positions are given.

Figure 3 shows a side view of the central β-barrel of AA2560 amylase oriented with the helix at the preceding amino acids 82-94. Residues at the bottom of the barrel are shown as spherical α-carbon positions and position numbers indicated.

Figure 4 is a cross-sectional view showing two helices in the AA2560 structural model. Positions 28 and 91 are shown as sticks. The left panel shows wild-type Arg28 and Ser91 in stick representation. The right panel shows the proximity positions of the two Arg residues in the S91R variant in stick representation.

Figure 5 is a table showing the performance of AA2560, AA560, AA707, and AAI10 combination variants in cleaning assays.

Detailed ways

Compositions and methods involving maltopentaose/maltohexaose variant amylases are described. Variants were discovered by experimental methods as detailed in the accompanying examples. Exemplary applications of variant amylases are for cleaning starch stains in dishwashing, laundry and other applications, for starch liquefaction or saccharification, for textile processing (eg desizing), in animal feed for improving digestibility , as well as for baking and brewing. These and other aspects of these compositions and methods are described in detail below.

Before describing various aspects and examples of the compositions and methods of the present invention, the following definitions and abbreviations are described.

1. Definitions and Abbreviations

In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a/an" and "the/the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "enzyme" includes a plurality of such enzymes, and reference to a "dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and the like.

This document is organized into sections for ease of reading; however, readers will appreciate that statements made in one section may apply to other sections. In this manner, headings used in different parts of this disclosure should not be construed as limiting.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For clarity, the following terms are defined below.

1.1. Abbreviations and Acronyms

Unless otherwise stated, the following abbreviations/acronyms have the following meanings:

°C Celsius

ADW automatic dishwashing

dH ₂ O or DI deionized water

_dIH2O deionized water, Milli-Q filtered

DNA deoxyribonucleic acid

EC Committee on Enzymes

g or gm grams

GA Glucoamylase

H ₂ O water

HDD Heavy Duty Powder Detergent

HDL High Density Liquid Detergent

hr hours

HSG high foam granular detergent

kDa kilodalton

kg kilogram

M mole

mg mg

min minutes

mL and ml

mm mm

mM Millimoles

MW molecular weight

MWU Modified Wohlgemuth Units; ^1.6x10-5 mg/MWU = Active Units

PI Performance Index

ppm parts per million, e.g. μg protein/gram dry solids

sec seconds

sp. species

U unit

v/v volume/volume

w/v weight/volume

w/w weight/weight

wt% weight percentage

μg microgram

μL and μl microliters

μm micrometer

μM micromolar

1.2. Definitions

The term "alpha-amylase" or "amylase" or generally amylase refers to an enzyme which, among other things, is capable of catalyzing the degradation of starch. Alpha-amylases are hydrolases that cleave α-D-(1→4)O-glycosidic bonds in starch. In general, alpha-amylases (EC 3.2.1.1; alpha-D-(1→4)-glucanoglucanohydrolases) are defined as endo-acting enzymes that cleave alpha within starch molecules in a random manner -D-(1→4)O-glycosidic linkages, resulting in polysaccharides containing three or more (1-4)-α-linked D-glucose units. In contrast, exo-acting amylolytic enzymes such as β-amylase (EC 3.2.1.2; α-D-(1→4)-glucanomaltohydrolase) and some product-specific α-amylases (eg Maltogenic alpha-amylase (EC 3.2.1.133)) cleaves polysaccharide molecules from the non-reducing end of a substrate. β-amylase, α-glucosidase (EC 3.2.1.20; α-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucan Glucoglucohydrolase) and product-specific amylases such as maltotetrasidases (EC 3.2.1.60) and maltohexasidases (EC 3.2.1.98) can produce malto-oligosaccharides of specific length or syrup-rich Specific malto-oligosaccharides. Some bacterial alpha-amylases primarily produce maltotetraose (G4), maltopentaose (G5) or maltohexaose (G6) from starch and related alpha-1,4-glucans, while most alpha-starches Enzymes further convert them into glucose and or maltose as final products. G6 amylases (such as AA560 amylase and Bacillus sp. 707 amylase derived from Bacillus sp. DSM 12649, the parent of STAINZYME ^™ ), also known as maltohexaose alpha-starch Enzyme (EC 3.2.1.98), technically exo-acting, but has a similar structure to alpha-amylases, and in some cases appears to respond to some of the same beneficial mutations.

An "enzyme unit" as used herein refers to the amount of product formed each time under specific assay conditions. For example, a "glucoamylase activity unit" (GAU) is defined as the amount of enzyme that produces 1 g of glucose per hour at 60°C, pH 4.2, from a soluble starch substrate (4% DS). A "Soluble Starch Unit" (SSU) is the amount of enzyme that produces 1 mg of glucose per minute from a soluble starch substrate (4% DS) at pH 4.5 at 50°C. DS stands for "Dry Solids"

The term "starch" refers to any material consisting of complex polysaccharide carbohydrates of plants consisting of amylose having the formula (C ₆ H ₁₀ O ₅ ) _x (where "X" can be any integer) and Made of amylopectin. The term includes plant-based materials such as grains, cereals, grasses, tubers and roots, and more particularly, from wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, milo, Material obtained from potato, sweet potato, and tapioca starch. The term "starch" includes granular starch. The term "granular starch" refers to raw starch (ie, uncooked starch), eg, starch that has not undergone gelatinization.

As used herein, the term "liquefaction" or "liquefaction" refers to the process of converting starch to lower viscosity and shorter chain dextrins.

With respect to a polypeptide, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polypeptide that does not contain artificial substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to a polynucleotide, the terms "wild-type", "parental" or "reference" refer to a naturally occurring polynucleotide that does not include artificial nucleoside changes. Note, however, that polynucleotides encoding wild-type, parental, or reference polypeptides are not limited to naturally occurring polynucleotides, and encompass any polynucleotides encoding wild-type, parental, or reference polypeptides.

References to wild-type polypeptides are understood to include mature forms of the polypeptides. A "mature" polypeptide or variant thereof is one in which a signal sequence is absent, eg, cleaved from an immature form of the polypeptide during or after expression of the polypeptide.

The term "variant" in reference to a polypeptide refers to a polypeptide that differs from the designated wild-type, parent or reference polypeptide in that it includes one or more naturally occurring or man-made amino acid substitutions, insertions or deletions. Similarly, the term "variant" in reference to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from the designated wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental or reference polypeptide or polynucleotide will be apparent from the context.

In the context of the alpha-amylases of the present invention, "activity" refers to alpha-amylase activity, which can be measured as described herein.

The term "performance benefit" refers to an improvement in a desired property of a molecule. Exemplary performance benefits include, but are not limited to: increased hydrolysis of starch substrates, enhanced liquefaction properties of grains, cereals or other starchy substrates, enhanced cleaning performance, enhanced thermal stability, enhanced detergent stability, enhanced storage stability, solubility Increased, pH profile altered, calcium-dependent decreased, specific activity increased, substrate specificity modified, substrate binding modified, pH-dependent activity modified, pH-dependent stability modified, oxidative stability increased, and expression Increase. In some cases, performance benefits are achieved at relatively lower temperatures. In some cases, performance benefits are realized at relatively higher temperatures.

The terms "protease" and "proteinase" refer to enzymatic proteins that have the ability to perform "proteolysis" or "proteolytic cleavage" which means that hydrolysis will form the A peptide bond that joins together amino acids in a peptide or polypeptide chain of a protein. This activity of a protease as a protein-digesting enzyme is referred to as "proteolytic activity".

The term "serine protease" refers to an enzyme that cleaves peptide bonds in proteins, wherein the enzyme serine acts as a nucleophilic amino acid at the enzyme active site. Serine proteases are divided into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. The most commonly used in laundry and dishwashing detergents are serine proteases, especially subtilisins.

A "combination variant" is a variant that includes two or more mutations, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, deletions, and/or insertions.

The term "recombinant," when used in reference to a subject cell, nucleic acid, protein, or vector, indicates that the subject has been modified from its natural state. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or express native genes at levels or under conditions different from those found in nature. The recombinant nucleic acid differs from the native sequence by one or more nucleotides, and/or is operably linked to a heterologous sequence, eg, a heterologous promoter in an expression vector. The recombinant protein can differ from the native sequence by one or more amino acids, and/or be fused to a heterologous sequence. A vector comprising a nucleic acid encoding an amylase is a recombinant vector.

The terms "recovered", "isolated" and "individually" refer to compounds, proteins (polypeptides), cells, nucleic acids, amino acids, such as naturally occurring, removed from at least one other material or component with which it is naturally associated. or other specified materials or components. An "isolated" polypeptide thereof includes, but is not limited to, a culture medium containing a secreted polypeptide expressed in a heterologous host cell.

The term "purified" refers to a material (eg, an isolated polypeptide or polynucleotide) in a relatively pure state, eg, at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

The term "enriched" refers to material (eg, isolated polypeptide or polynucleotide) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

The terms "thermostable" and "thermostability" in reference to an enzyme refer to the ability of an enzyme to remain active after exposure to elevated temperatures. The thermostability of an enzyme (eg amylase) is measured by its half-life (t1/2), given in minutes, hours or days, during which half of the enzyme activity is lost under defined conditions. Half-life can be calculated by measuring residual alpha-amylase activity after exposure (ie, challenged) to elevated temperatures.

A "pH range" in reference to an enzyme refers to the range of pH values at which the enzyme exhibits catalytic activity.

The terms "pH stability" and "pH stability" in relation to enzymes relate to the ability of an enzyme to maintain activity for a predetermined period of time (eg, 15 min., 30 min., 1 hour) over a wide range of pH values.

The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein" and "peptide" and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". Amino acid sequences are expressed in standard amino-to-carboxy-terminal orientation (ie, N→C) using conventional one-letter or three-letter codes for amino acid residues.

The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded, and can contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences encoding particular amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

"Synthetic" molecules are produced by chemical or enzymatic synthesis in vitro rather than by an organism.

In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection", "transformation" or "transduction" as known in the art.

A "host strain" or "host cell" is an organism into which an expression vector, bacteriophage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (eg, an amylase), has been introduced. Exemplary host strains are microbial cells (eg, bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and/or fermenting carbohydrates. The term "host cell" includes protoplasts produced from the cell.

The term "heterologous" in reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

The term "endogenous" in reference to a polynucleotide or protein refers to a polynucleotide or protein that is naturally present in a host cell.

The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. This process includes both transcription and translation.

A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates extracellular secretion of the protein. The mature form of the extracellular protein lacks the signal sequence that is cleaved during secretion.

"Biologically active" refers to a sequence having the specified biological activity, eg, enzymatic activity.

The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is usually expressed as units (U)/mg protein.

As used herein, "water hardness" is a measure of the presence of minerals (eg, calcium and magnesium) in water.

"Amylase-containing cultured cell material" or similar language refers to a cell lysate or supernatant (including medium) that includes amylase as a component. The cellular material can be from a heterologous host, which is grown in culture for the purpose of producing amylase.

"Percent sequence identity" means that a particular sequence has at least a percentage of amino acid residues that are identical to amino acid residues in a given reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. [Nucleic Acids Research] 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

Deletions are considered non-identical residues compared to the reference sequence.

The term "dry solids content" (ds) refers to the total solids of the slurry on a dry weight percent basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

The phrase "simultaneous saccharification and fermentation (SSF)" refers to a biochemical production process in which a microorganism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present in the same process step. SSF involves the simultaneous hydrolysis of starch substrates (granulated, liquefied or dissolved) to sugars (including glucose) and the fermentation of sugars to alcohols or other biochemicals or biological materials in the same reaction vessel.

"Ethanologenic microorganism" refers to a microorganism having the ability to convert sugars or oligosaccharides to ethanol.

The term "fermented beverage" refers to any beverage produced by methods including fermentation processes (eg, microbial fermentation, such as bacterial and/or fungal fermentation).

The term "malt" refers to any malted cereal grain, such as malted barley or wheat.

The term "mash" refers to an aqueous slurry of any starch and/or sugar-containing vegetable material such as grain flour (e.g. including crushed barley malt, crushed barley) and/or other adjuvants or combinations thereof, subsequently mixed with water. Blend to separate into wort and spent grains.

The term "wort" refers to the unfermented liquid run-off after extraction of the flour during mash preparation.

The term "about" refers to ±15% of the reference value.

2. Maltopentaose/maltohexaose alpha-amylase variants

Combination variants of maltopentaose/maltohexaose alpha-amylases are described which show a high degree of performance in automatic dishwashing (ADW) applications. These variants are most closely related to an alpha-amylase from Bacillus sp. (referred to herein as AA2560 and previously identified in WO 2018/184004 as BspAmy24 (SEQ ID NO: 1)). The mature amino acid sequence of AA2560 alpha-amylase is shown in the following SEQ ID NO: 1:

A closely related maltopentaose/maltohexaose alpha-amylase is from Bacillus sp. 707, referred to herein as "AA707". The mature amino acid sequence of AA707α- is shown in the following SEQ ID NO:2:

Another closely related maltopentaose/maltohexaose alpha-amylase is from Bacillus sp., designated AA560. The mature amino acid sequence of AA560 is shown in SEQ ID NO: 3 below:

Based on amino acid sequence identity, another putative maltopentaose/maltohexaose alpha-amylase is from another Bacillus species and is referred to herein as AAI10. The mature amino acid sequence of AAI10 alpha-amylase is shown in the following SEQ ID NO:4:

An alignment of these four alpha-amylases is shown in Figure 1 . Amino acid sequence identities are summarized in Table 1. AA707, AA560 and AAI10 all share greater than 80% amino acids with AA2560.

Table 1. Amino acid sequence identity of alpha-amylases

A feature of the variants of the invention is the mutation at position 91 and/or at least one mutation at the bottom of the alpha-amylase TIM barrel structure. Residues at the bottom of the barrel have a solvent accessible surface area greater than zero and are located at or adjacent to the core β-barrel structure, on the side of the barrel opposite the active site, and on the side containing the N-terminus of each chain. Solvent-accessible surface areas were calculated using MOE 2018.01 (Chemical Computing Group, Montreal), using default parameters, and based on the AA2560 homology model constructed using MOE 2018.01 using default parameters and the 1BLI structure from pdb. The relevant residues are located at positions 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, numbered with reference to SEQ ID NO: 1, 325, 362, 363, and 364. The structural significance of these barrel bottom residues can be understood with the help of the images in Figures 2 and 3. In all cases, the residues are arranged at the bottom of the TIM barrel structure, which represents a major structural feature of alpha-amylase and many other enzymes. An exemplary mutation at residue 91 is the substitution of a polar residue with a charged residue, particularly a positively charged residue (eg, arginine (ie, X91R)), which is a specific substitution in the case of AA2560 S91R.

A notable exception is that Amy707 differs from AA2560, AAI10, and AA560 (and from Cytophaga sp. (Jeang, C-L. et al. (2002) Applied and Environmental Microbiolgy), 68:3651-54; Genbank Accession No. AAA22231), Bacillus sp. TS-23 (Lin, L-L. et al. (1997) J Appl Microbiol, 82:325-34; Genbank Accession No. AAA63900) and other amylases) because it does not have a positively charged amino acid residue at position 28. AA2560, AAI10, and AA560, numbered with reference to SEQ ID NO: 1, have an Arg or His side chain (position 28), while Amy707 has an Asn, which cannot be positively charged (Table 2).

Table 2. Amino acid side chains at position 28 of the four amylases

amylase Wild type residue at position 28 AA707 Asn AA2560 Arg AAl10 His AA560 Arg

Thus, in AA2560, AA560, AAI10, and many other amylases, mutation of Ser91 to Arg will result in two positively charged amino acids in very close proximity, as is evident from the model of AA2560 shown in Figure 4 . Without being bound by theory, it is hypothesized that the proximity of these residues may beneficially affect many amylases such as AA2560 by repositioning the helix at positions 22-37 and 82-95 (as a result of charge-charge repulsion) ) activity. In contrast, Asn28 in wild-type Amy707 may be able to form a hydrogen bond with Arg at position 91, which may lead to a tighter interaction (possibly detrimental to the activity of Amy707). As above and below, SEQ ID NO: 1 is used for numbering.

Exemplary mutations of barrel bottom residues are substitutions including, but not limited to, the following: X40N, X40D, X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L, and X364M, where "X" is the previous Amino acid residues present in the wild-type parental alpha-amylase. The specific mutations referenced to AA2560 are T40N, T40D, Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L, and Y364M.

Described differently, variants have one, two, three, or more features including: N or D at position 40, F or L at position 100, Y at position 263, D, K or Q, R, N or M at position 324, or L or M at position 364.

While the mutation at position 91 and the combination of mutations at the bottom of the barrel produced superior performance advantages, each mutation alone appeared to yield benefits, and some of the variants of the invention had mutations at only one position/structure.

Variants may additionally have characteristic mutations in a loop comprising surface exposed residues 167, 169, 171, 172, and 176 numbered with reference to SEQ ID NO:1. Exemplary mutations include, but are not limited to, the substitutions: X167F, X169H, X171Y, X172R, X172N, and X176S, and specifically W167F, Q169H, R171Y, Q172R, Q172N, and R176S. Described differently, variant feature substitutions include F at position 167, H at position 169, Y at position 171, R or N at position 172, and/or position 176 numbered with reference to SEQ ID NO: 1 S at the place.

Variants may additionally have characteristic mutations at positions 116 and 281 that are believed to affect solubility. Exemplary mutations at these positions are the substitutions X116R and X281S, specifically the substitutions W116R and H281S.

Variants may additionally have characteristic stabilizing mutations at positions 190 and/or 244 numbered with reference to SEQ ID NO:1. Such mutations are well classified and included in currently commercially available alpha-amylases (for cleaning, grain processing and textile processing). Exemplary mutations in these residues are the substitutions X190P and X244A, E or Q, particularly E190P, S244A, S244E, and S244Q. The combination of mutations at positions 275 and 279 with the mutation at position 190 is also of interest.

Variants may additionally have characteristic mutations at positions 1, 7, 118, 195, 202, 206, 321, 245, and 459 numbered with reference to SEQ ID NO: 1, and these variants are included in the currently commercially available Alpha-amylases are or have been proposed for such applications.

Variants may further include deletions in the _X1G / _X2G2 motif adjacent to the calcium-binding loops corresponding to R181, G182, T183, and G184 numbered using SEQ ID NO: ₁ . In some embodiments, variant alpha-amylases include adjacent, pairwise deletions of amino acid residues corresponding to R181 and G182, or T183 and G184. Deletions in amino acid residues corresponding to R181 and G182 may be referred to as "ΔRG", while deletions of amino acid residues corresponding to residues at position 183 (usually T, D, or H) and G184 may be appropriately referred to as It is "ΔTG", "ΔDG", "ΔHG", etc. Both pairwise deletions appear to have the same effect in alpha-amylase.

Variants may further include previously described mutations for use in other alpha-amylases with (i) well-known Bacillus alpha-amylases (eg, from B. lichenifomis) (i.e. BLA and LAT), B. stearothermophilus (i.e. BSG) and B. amyloliquifaciens (i.e. P00692, BACAM and BAA)) or hybrids thereof, ( ii) any alpha-amylase classified as a carbohydrate-active enzyme database (CAZy) family 13 alpha-amylase, or (iii) any amylase previously mentioned with the descriptive term "Termamyl-like" having a similar fold and/or have 60% or more amino acid sequence identity. Exemplary alpha-amylases include, but are not limited to, those from Bacillus sp. SG-1, Bacillus sp. 707, and alpha-amylases designated A7-7, SP722, DSM90 14, and KSM AP1378. Similarly, any combination of the mutations described herein can yield performance advantages in these alpha-amylases, whether or not they are described as maltopentaose/maltohexaose alpha-amylases.

Specific contemplated combinatorial variants are listed below (numbering using SEQ ID NO: 1). As mentioned above, similar variants with ΔR183-ΔT184 rather than ΔR181-ΔG182 are expected to perform as well as those described in detail.

T40-S91-Q169-ΔR181-ΔG182-T183-H281

Q172-ΔR181-ΔG182-E190-S288

Q172-ΔR181-ΔG182-S244-S288-S474

S91-Q172-ΔR181-ΔG182-E190-I324

T40-S91-ΔR181-ΔG182-E190-F263

T40-S91-ΔR181-ΔG182-S244-Y364

S91-Q172-ΔR181-ΔG182-E190-I324

S91-W116-Q172-ΔR181-ΔG182-S244-H281-S288

T40-S91-Y100-W116-Q172-ΔR181-ΔG182-S244-H281

T40-S91-Q172-ΔR181-ΔG182-S244-F263-H281

S91-Q172-ΔR181-ΔG182-E190-I324

T40-S91-Q172-ΔR181-ΔG182-E190-H281-I324

Y364-ΔR181-ΔG182

In related alpha-amylases (including previously engineered alpha-amylases), mutations can be described as:

X40-X91-X169-ΔR181-ΔG182-X183-X281

X172-ΔR181-ΔG182-X190-X288

X172-ΔR181-ΔG182-X244-X288-X474

X91-X172-ΔR181-ΔG182-X190-X324

X40-X91-ΔR181-ΔG182-X190-X263

X40-X91-ΔR181-ΔG182-X244-X364

X91-X172-ΔR181-ΔG182-X190-X324

X91-X116-X172-ΔR181-ΔG182-X244-X281-X288

X40-X91-X100-X116-X172-ΔR181-ΔG182-X244-X281

X40-X91-X172-ΔR181-ΔG182-X244-X263-X281

X91-X172-ΔR181-ΔG182-X190-X324

X40-X91-X172-ΔR181-ΔG182-X190-X281-X324

X364L-ΔR181-ΔG182

Such variants include those having two, three, four, five, six or more of the following characteristics: (a) D or N at position 40 and/or R at position 91 and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and/or L at position 364, optionally with (c) position 169 H at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R at position 474, position 116 A combination of R at , optionally with paired deletions at positions 181 and 182 or 183 and 184.

The specific substitutions in the tested variants are listed below:

T40N-S91R-Q169H-ΔR181-ΔG182-T183M-H281N

Q172R-ΔR181-ΔG182-E190P-S288D

Q172R-ΔR181-ΔG182-S244E-S288D-S474R

S91R-Q172R-ΔR181-ΔG182-E190P-I324M

T40N-S91R-ΔR181-ΔG182-E190P-F263Y

T40N-S91R-ΔR181-ΔG182-S244E-Y364L

S91R-Q172R-ΔR181-ΔG182-E190P-I324R

S91R-W116R-Q172R-ΔR181-ΔG182-S244E-H281S-S288D

T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S

T40N-S91R-Q172R-ΔR181-ΔG182-S244Q-F263Y-H281S

S91R-Q172R-ΔR181-ΔG182-E190P-I324N

T40D-S91R-Q172R-ΔR181-ΔG182-E190P-H281S-I324R

Y364L-ΔR181-ΔG182

It will be appreciated that when the alpha-amylase naturally possesses the mutations listed above (ie, wherein the wild-type alpha-amylase already contains the residues identified as mutated), then that particular mutation is not applicable to the molecule. However, other described mutations can act in combination with naturally occurring residues at this position.

The variant alpha-amylases of the invention may also include substitutions, deletions or additions of one or several amino acids in the amino acid sequence (e.g. less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3 or even less than 2 substitutions, deletions or additions). Such variants are expected to have similar activity to the alpha-amylase from which they are derived. The variant alpha-amylases of the invention may also include minor deletions and/or extensions of one or several residues at their N- or C-terminus. Such minor changes are unlikely to destroy the inventive concepts described herein.

The amylases of the invention may be "precursor", "immature" or "full length", in which case they contain a signal sequence; or "mature", in which case they lack a signal sequence . The mature form of the polypeptide is usually the most useful. Unless otherwise indicated, amino acid residue numbering as used herein refers to the mature form of the corresponding amylase polypeptide.

In some embodiments, the variant alpha-amylase is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90% of SEQ ID NO: 1, 2, 3, or 4. At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100% amino acid sequence identity.

2.5. Nucleotides encoding variant amylase polypeptides

In another aspect, nucleic acids encoding variant alpha-amylase polypeptides are provided. The nucleic acid may encode a specific amylase polypeptide, or an alpha-amylase having a specified degree of amino acid sequence identity with a specific alpha-amylase.

In some embodiments, the nucleic acid encodes an alpha-amylase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100% identical in amino acid sequence sex. It will be appreciated that due to the degeneracy of the genetic code, multiple nucleic acids can encode the same polypeptide.

In some embodiments, the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid encoding an alpha amylase (or a nucleic acid complementary to a nucleic acid encoding an alpha amylase), the alpha-amylase and SEQ ID NOs: 1, 2 , 3, or 4 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, or even at least 99%, but less than 100% amino acid sequence identity.

3. Production of Variant Alpha-Amylases

The variant alpha-amylases of the invention can be produced in host cells using methods well known in the art, eg, by secretion or intracellular expression. Fermentation, isolation and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated solutions containing variant alpha-amylase polypeptides.

For production-scale recovery, variant alpha-amylase polypeptides can be enriched or partially purified by removing cells by flocculation with polymers as described in the general case above. Alternatively, the enzymes can be enriched or purified by microfiltration and then concentrated by ultrafiltration using available membranes and equipment. However, for some applications, the enzymes do not require enrichment or purification, and whole broth cultures can be lysed and used without further processing. The enzymes can then be processed into particles, for example.

4. Cleaning compositions containing variant alpha-amylases

One aspect of the compositions and methods of the present invention pertains to a cleaning composition comprising modified alpha-starch as a component for applications such as automatic and manual dishwashing (ADW), laundry and other hard surface cleaning enzymes.

4.1. Overview

Preferably, the variant alpha-amylase is incorporated into detergent formulations at or below concentrations conventionally used for known alpha-amylases. Because the alpha-amylase variant outperforms any previously available alpha-amylase, it is expected to provide superior performance at standard doses and similar performance at lower doses compared to existing alpha-amylases performance. Specific forms and formulations for detergent compositions comprising the alpha-amylases of the present invention are described below.

4.2. Automatic dishwashing (ADW) detergent compositions

Exemplary ADW detergent compositions include nonionic surfactants including ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy terminated poly(oxyalkylated) alcohols or amine oxides surfactants, which are present in amounts from 0% to 10% by weight; builders (builders in the range of 5%-60%) include: phosphate builders (eg monophosphates, diphosphates salts, tripolyphosphates, other oligophosphates, sodium tripolyphosphate-STPP), and phosphate-free builders (such as amino acid-based compounds including methyl-glycine-diacetic acid (MGDA) and its salts and Derivatives, glutamic acid-N,N-diacetic acid (GLDA) and its salts and derivatives, iminodisuccinic acid (IDS) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives, Nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), beta-alanine diacetic acid (beta-ADA) and its salts), polycarboxylic acids and their partially or fully neutralized salts Homopolymers and copolymers, monomeric polycarboxylic and hydroxycarboxylic acids and salts thereof, in the range of 0.5% to 50% by weight; sulfonated/in the range of about 0.1% to about 50% by weight Carboxylated polymers to provide dimensional stability; drying aids (e.g., polyesters, especially anionic polyesters (optionally combined with 3 to 6 polycondensation-facilitating polymers) in the range of about 0.1% to about 10% by weight functional groups - typically acid, alcohol or ester functional groups together with additional monomers), polycarbonate-, polyurethane- and/or polyurea-polyorganosiloxane compounds or precursor compounds thereof, especially reactive cyclic carbonic acids silicates (including sodium or potassium silicates such as sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) in the range of about 1% to about 20% by weight ; inorganic bleaches (eg, peroxyhydrate salts such as perborates, percarbonates, perphosphates, persulfates, and persilicates) and organic bleaches (eg, organic peroxyacids, including diacyl and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); bleach activators (i.e. organic peracid precursors, which in the range of about 0.1% to about 10% by weight); bleach catalysts (eg, manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamines and related complexes, and pentapyridine Cobalt(III) amine acetate and related complexes); metal conditioners (eg, benzotriazoles, metal salts and complexes, and/or silicates) in the range of about 0.1% to 5% by weight ); enzymes ranging from about 0.01 mg to 5.0 mg active enzymes per gram of automatic dishwashing detergent composition (eg protease, alpha-amylase, lipase, cellulase, choline oxidase, peroxidase/ oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and mixtures thereof); and enzyme stabilizers Components (eg, oligosaccharides, polysaccharides, and inorganic divalent metal salts).

Specific exemplary ADW compositions are shown in Table 2, in which at least some of the present variants have been tested.

Table 2. Exemplary ADW Compositions

4.3. Heavy Duty Liquid (HDL) Laundry Detergent Compositions

Exemplary HDL laundry detergent compositions include detersive surfactants (10% to 40% wt/wt), including anionic cleaning surfactants (selected from the group of linear or branched or random chain, substituted or un Substituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and/or mixtures thereof) and optionally nonionic Surfactants (selected from the group: linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8-C18 alkyl ethoxylated alcohols and/or C6-C12 alkyl phenol alkoxylates) wherein the weight ratio of anionic cleaning surfactant (having a hydrophilic index (HIc) from 6 to 9) to nonionic cleaning surfactant is greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from the group consisting of hydrocarbyl pyridinium compounds, hydrocarbyl quaternary ammonium compounds, hydrocarbyl quaternary phosphonium compounds, hydrocarbyl tertiary sulfonium compounds, and/or mixtures thereof); amphoteric Ionic and/or amphoteric detersive surfactants (selected from the group: alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof.

The composition may optionally include a surface-active enhancing polymer consisting of amphiphilic alkoxylated grease cleaning polymers (selected from the group consisting of branched hydrophilic and hydrophobic Characteristic alkoxylated polymers, such as alkoxylated polyalkyleneimines (in the range of 0.05 wt % to 10 wt %) and/or random graft polymers (typically containing a compound selected from the group consisting of: The hydrophilic backbone of the group of monomers: unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols (such as glycerol) and mixtures thereof); and one or more hydrophobic side chains selected from the group consisting of C4-C25 alkyl groups, polypropylene, polybutene, ethylene of saturated C1-C6 monocarboxylic acids C1-C6 alkyl esters of acrylic or methacrylic acid and mixtures thereof.

The composition may include additional polymers such as soil release polymers (including anionically terminated polyesters (eg SRP1); polymers comprising at least one monomeric unit (in random or block configuration), the monomeric The body units are selected from sugars, dicarboxylic acids, polyols and combinations thereof; ethylene glycol terephthalate based polymers and their copolymers (in random or block configuration) such as Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL); anti-redeposition polymers (0.1 wt % to 10 wt %, including carboxylate polymers, if containing at least A polymer of monomers selected from the group consisting of acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylene malonate Acids and any mixtures thereof; vinylpyrrolidone homopolymers; and/or polyethylene glycols ranging in molecular weight from 500 to 100,000 Da); cellulose polymers (including those selected from the group consisting of alkyl cellulose, alkyl alkoxy Cellulose polymers of alkyl cellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose methylcellulose and mixtures thereof) and polymeric carboxylates such as maleate/acrylate random copolymers or polyacrylate homopolymers.

The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C12-C24 fatty acids (0 to 10 wt%); deposition aids (examples of which include polysaccharides; preferably cellulose polymers; DADMAC); and copolymers of DAD MAC with vinylpyrrolidone, acrylamide, imidazole, imidazoline halides, and mixtures thereof (in random or block configurations); cationic guar; cationic cellulose , such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide, and mixtures thereof.

The composition may further comprise dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, N-vinylpyrrolidone and N-vinylimidazole Copolymers, polyvinyloxazolidinone and polyvinylimidazole and/or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentamethylenephosphonic acid ( DTPMP), hydroxyethanediphosphonic acid (HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), diethylenetriaminepentaacetic acid (DTPA), propylene Diaminetetraacetic acid (PDTA), 2-hydroxypyridine-N-oxide (HPNO), or methylglycine diacetic acid (MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamic acid) Amino acid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA), 4,5-dihydroxyisobenzenedisulfonic acid, citric acid and any salt thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA) , Triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof.

The composition preferably includes an enzyme selected from the group consisting of (typically about 0.01 wt% active enzyme to 0.03 wt% active enzyme): protease, alpha-amylase, lipase, cellulase, choline oxidase, peroxidase /oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixtures thereof. The composition may include an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol; sugars or sugar alcohols; lactic acid; reversible protease inhibitors; boronic acids or boronic acid derivatives such as aromatic boronic esters; or phenylboronic acid derivatives compounds such as 4-formylphenylboronic acid).

The composition optionally includes a silicone or fatty acid based suds suppressor; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti-foaming agents (0.001 wt% to about 4.0 wt%) and/or structurants/ Thickener (0.01 wt% to 5 wt% selected from the group consisting of di- and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, microcellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).

The composition may be in any liquid form, such as liquid or gel form, or any combination thereof. The composition may be in any unit dosage form, eg, a sachet.

4.4. Heavy Duty Dry/Solid (HDD) Laundry Detergent Compositions

Exemplary HDD laundry detergent compositions include detersive surfactants, including anionic detersive surfactants (eg, linear or branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonic acids) salts, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof); nonionic detersive surfactants (eg, linear or branched chain or Random chain, substituted or unsubstituted C8-C18 alkyl ethoxylates and/or C6-C12 alkyl phenol alkoxylates); cationic detersive surfactants (eg, alkyl pyridine compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulfonium compounds and mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (eg, alkanolamine sulfobetaines), amphoteric surfactants, Semi-polar nonionic surfactants; and mixtures thereof; builders, including phosphate-free builders (such as zeolite builders, examples of which include zeolite A, zeolite X, Zeolite P and Zeolite MAP), phosphate builders (eg sodium tripolyphosphate in the range from 0 wt% to less than 10 wt%), citric acid, citrates and nitrilotriacetic acid, silicates (eg, in the Sodium silicate or potassium silicate or sodium metasilicate, or layered silicate (SKS-6) in the range from 0 wt% to less than 10 wt%; carbonates (eg, in the range from 0 wt% to less than 80 wt%) sodium carbonate and/or sodium bicarbonate); and bleaching agents, including photobleaches (eg, sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes, and mixtures thereof); Bleach activators (e.g. dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate acid salts, tetraacetylethylenediamine-TAED, nonanoyloxybenzenesulfonate-NOBS, nitrile quaternary ammonium salts, and mixtures thereof); sources of hydrogen peroxide (eg, inorganic peroxyhydrate salts, examples of which include mono- or tetrahydrate sodium salts of borates, percarbonates, persulfates, perphosphates or persilicates); preformed hydrophilic and/or hydrophobic peracids (eg percarboxylic acids and salts, per carbonic acid and salts, periodic acid and salts, peroxymonosulfuric acid and salts, and mixtures thereof); and/or bleach catalysts (eg, imine bleach accelerators, examples of which include imine cations and polyions, imide zwitterions , modified amine, modified amine oxide, N-sulfonyl imine, N-phosphono imide, N-acyl imide, thiadiazole dioxide, perfluoroimine, cyclic sugar ketone and mixtures thereof) ; and metal-containing bleach catalysts (e.g. copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations and auxiliary metal cations (e.g. zinc or aluminium) and chelates (e.g. EDTA, EDTA) methylenephosphonic acid) and its water-soluble salts)).

The composition preferably includes enzymes such as protease, alpha-amylase, lipase, cellulase, choline oxidase, peroxidase/oxidase, pectate lyase, mannanase, cutinase, Laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixtures thereof.

The composition may optionally include additional detergent ingredients including perfume microcapsules, starch encapsulated perfume coordinators, toners, additional polymers (including fabric integrity and cationic polymers), dye locking ingredients , fabric softeners, brighteners (eg C.I. optical brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids and/or cyclodextrins.

4.5. Additional enzymes

Any of the cleaning compositions described herein can include any number of additional enzymes. Typically, the one or more enzymes should be compatible with the selected detergent (eg, in terms of pH optimization, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the one or more enzymes should be Present in an effective amount. The following enzymes are provided as examples.

Protease:

Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are included, as well as naturally processed proteins. The protease may be a serine or metalloprotease, an alkaline microbial protease, a trypsin-like protease, or a chymotrypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus species, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (see e.g. , WO 1989/06279). Exemplary proteases include, but are not limited to, WO 1995/23221, WO 1992/21760, WO2008/010925, WO 2010/0566356, WO 2011/072099, WO 2011/13022, WO 2011/140364, WO2012/151534, WO 2015/038 WO 2015/089441, WO 2015/089447, WO 2015/143360, WO2016/001449, WO 2016/001450, WO 2016/061438, WO 2016/069544, WO 2016/069548, WO2016/06965572, WO2016/0696557 /069563, WO 2016/069569, WO 2016/087617, WO2016/087619, WO 2016/145428, WO 2016/174234, WO 2016/183509, WO 2016/202835, WO2016/205755, WO 2508/011 、WO 2018/169750、WO/2018/118917、US5,801,039、US 5,340,735、US 5,500,364、US 5,855,625、RE 34606、US 5,955,340、US 5,700,676、US 6,312,936、US 6,482,628、US 8,530,219中描述的那些；以及WO 2007 Metalloproteases described in WO 2014/194032, WO 2014/194034, WO 2014/194054 and WO 2014/194117.

OXP、

PREFERENZ^TM蛋白酶(例如P100、P110、P280、P300)、EFFECTENZ^TM蛋白酶(例如P1000、P1050、P2000)、EXCELLENZ^TM蛋白酶(例如P1000)、

和PURAFAST(美国丹尼斯科公司(Danisco US))；

ULTRA、

16L、

ULTRA、

PRIMASE、DURAZYM、

PROGRESS

和

(诺维信公司(Novozymes))；BLAP^TM和BLAP^TM变体(汉高公司(Henkel))；LAVERGY^TM PRO 104L(BASF)、和

(嗜碱芽孢杆菌(B.alkalophilus)枯草杆菌蛋白酶(花王公司(Kao)))。适合的蛋白酶包括特别选择或工程化以在相对低温下起作用的天然存在的蛋白酶或者工程化变体。Exemplary commercial proteases include, but are not limited to, MAXATASE, MAXACAL, MAXAPEM,

OXP,

PREFERENZ ^™ proteases (eg P100, P110, P280, P300), EFFECTENZ ^™ proteases (eg P1000, P1050, P2000), EXCELLENZ ^™ proteases (eg P1000),

and PURAFAST (Danisco US);

ULTRA,

16L,

ULTRA,

PRIMASE, DURAZYM,

PROGRESS

and

(Novozymes); BLAP ^™ and BLAP ^™ variants (Henkel); LAVERGY ^™ PRO 104L (BASF), and

(B. alkalophilus subtilisin (Kao)). Suitable proteases include naturally occurring proteases or engineered variants that are specifically selected or engineered to function at relatively low temperatures.

In particular embodiments of the compositions and methods of the present invention, the alpha-amylase variant is used in combination with a variant subtilisin from Bacillus gibii (referred to as BG46) having amino acid substitutions X39E, X99R, X126A, X127E, and X128G, and further have one or more additional substitutions selected from the group consisting of: N74D-M211L-N253P, R179Q-M211L-N253P, N74D-N253P, N85R-G160Q-R179Q -M211L-N212S-N253P、R179Q-N253P、G160Q-R179Q-M211L-N212S-N253P、R179Q-M211L、G160Q-R179Q-M211L-N253P、G160Q-R179Q-N212S-N253P、N74D-M211L、M211L-N242D、G160Q -R179Q-M211L-N212S, N74D-R179Q-M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N74D-Q200L-M211L, N74D-G160Q-N212S-N253P, N760Q-N253P -R179Q, G160Q-R179Q-N212S, N74D-G160Q-N253P, N74D-G160Q-R179Q-M211L-N212S-N253P, N74D-N085R-G160Q-R179Q-M211L, N74D-G160Q-M211L-N74-N212 -N116R-Q200L-Q256E, N74D-G160Q-R179Q-N212S-N253P, N74D-G160Q-M211L-N212S, N74D-G160Q, N74D-G160Q-R179Q-M211L-N253P, N74D-R179Q-M211L-N74D-S , N74D-G160Q-M211L, N74D-G160Q-R179Q-N253P, N74D, N74D-G160Q-R179Q-M211L-N212S, N74D-N085R-M211L-N212S, N74D-G160Q-R179Q-N216L-R179Q-N212S, N74D-G16Q-R174D , N74D-M211L-Q256E, N74D-G160Q-R179Q, R179Q-M211L-N212S-N253P, R179Q-M211L-N212S, N 74D-N085R-R179Q-M211L-N212S, N74D-M211L-N212S, N74D-R179Q-M211L-N212S, N74D-M211L-N242D, N74D-Q200L-M211L-Q256E, N74D-N2-Q2-M2511L-N74D-Q200Q2-M2511L- N74D-M211N-N212Q N74D-N198A-M211Q-N212Q N198L-M211Q-N212Q, N74D-N198Q-M211Q-N212Q, N74D-N198R-M211Q-N212Q, N74D-N198T-M211Q-N212Q, N74D-N198V-M211Q-N212Q, N74D-N212Q-Q256E, N74D-N212Q-Q256E R207Q, N74D-R207Q-M211N, N74D-R207Q-M211N-N212Q, N74D-R207Q-M211N-N212Q-Q256E, N74D-R207Q-M211N-Q256E, N74D-R207Q-M211Q, N74Q-R221N-N212Q-Q207Q- R207Q-M211Q-N212Q-Q256E, N74D-R207Q-N212Q, N74D-R207Q-N212Q-Q256E, N74D-R207Q-Q256E, N74D-N198S-M211Q, and N74D-N198L-M211Q, where the amino acid positions by corresponding to SEQ ID NO The amino acid sequence of SEQ ID NO:5 is numbered, wherein the variant has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:6. These amino acid sequences are shown as follows:

Amino acid sequence of BG46 protease (SEQ ID NO: 5):

QQTVPWGITRVQAPAVHNRGITGSGVRVAILDSGISAHSDLNIRGGASFVPGEPTTADLNGHGTHVAGTVAALNNSIGVIGVAPNAELYAVKVLGANGSGSVSGIAQGLEWAATNNMHIANMSLGSDFPSSTLERAVNYATSRDVLVIAATGNNGSGSVGYPARYANAMAVGATDQNNRRANFSQYGTGIDIVAPGVNVTRQSTYPGNRYVSMNGTSMATPHVAGAAALVKQRYPSFGSGLVNATQIRNANHATLQ

Amino acid sequence of BG46 with substitutions S39E, S99R, S126A, D127E, and F128G (SEQ ID NO:6):

QQTVPWGITRVQAPAVHNRGITGSGVRVAILDSGISAHEDLNIRGGASFVPGEPTTADLNGHGTHVAGTVAALNNSIGVIGVAPNAELYAVKVLGANGRGSVSGIAQGLEWAATNNMHIANMSLGAEGPSSTLERAVNYATSRDVLVIAATGNNGSGSVGYPARYANAMAVGATDQNNRRANFSQYGTGIDIVAPGVNVTRQSTYPGNRYVSMNGTSMATPHVAGAAALVKQRYPSFGSGLVNATQIRNHANATQ

Lipase:

Suitable lipases include those of bacterial or fungal origin. Chemically modified, proteolytically modified or protein engineered mutants are included. Examples of useful lipases include, but are not limited to, lipases from Humicola (syn. Thermomyces sp.), such as from H. lanuginosa (thermophilus lanuginosa). T. lanuginosus) (see eg EP 258068 and EP 305216), from H. insolens (see eg WO 96/13580); Pseudomonas lipase ( For example, from P. alcaligenes or P. pseudoalcaligenes; see eg, EP 218 272); P. cepacia (see eg, EP 331 376); P. stutzeri (see eg, GB 1,372,034); P. fluorescens; Pseudomonas sp. strain SD 705 (see eg, WO 95/06720 and WO 96/27002); P. wisconsinensis (see eg WO 96/12012); Bacillus lipase (eg from Bacillus subtilis; see eg Dartois et al (1993) ), Biochemica et Biophysica Acta [Acta Biochemistry and Biophysics], 1131:253-360); Bacillus stearothermophilus (see, e.g., JP 64/744992); or B. pumilus (see, e.g., WO 91/16422). Additional lipase variants contemplated for use in formulations include, for example, WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO Those described in 95/22615, WO 97/04079, WO 97/07202, EP 407225 and EP 260105.

和

ULTRA(诺维信公司)；和LIPASE P(天野药业有限公司(Amano Pharmaceutical Co.Ltd))。Exemplary commercial lipases include, but are not limited to, M1 LIPASE, LUMA FAST, and LIPOMAX (Genencor);

and

ULTRA (Novozymes Corporation); and LIPASE P (Amano Pharmaceutical Co. Ltd).

Polyesterase:

Suitable polyesterases may be included in the compositions, such as those described, for example, in WO 01/34899, WO 01/14629, and US 6,933,140.

Amylase:

和BAN^TM(诺和诺德公司(Novo Nordisk A/S)和诺维信公司)；

和PREFERENZ^TM(来自杜邦工业生物科学公司(DuPont Industrial Biosciences))。示例性α-淀粉酶描述于WO 94/18314 A1、WO 2008/0293607、WO 2013/063460、WO 10/115028、WO 2009/061380 A2、WO 2014/099523、WO 2015/077126 A1、WO 2013/184577、WO 2014/164777、W0 95/10603、WO 95/26397、WO 96/23874、WO 96/23873、WO 97/41213、WO 99/19467、WO 00/60060、WO 00/29560、WO 99/23211、WO99/46399、WO 00/60058、WO 00/60059、WO 99/42567、WO 01/14532、WO 02/092797、WO 01/66712、WO 01/88107、WO 01/96537、WO 02/10355、WO 2006/002643、WO 2004/055178、和WO98/13481中。The compositions of the present invention can be combined with other amylases, including other alpha-amylases. This combination is particularly desirable when different alpha-amylases exhibit different performance characteristics and the combination of multiple different alpha-amylases results in a composition that provides the benefits of the different alpha-amylases. Other alpha-amylases include commercially available alpha-amylases such as but not limited to

and BAN ^TM (Novo Nordisk A/S and Novozymes);

and PREFERENZ ^™ (from DuPont Industrial Biosciences). Exemplary alpha-amylases are described in WO 94/18314 A1, WO 2008/0293607, WO 2013/063460, WO 10/115028, WO 2009/061380 A2, WO 2014/099523, WO 2015/077126 A1, WO 2013/184577 , WO 2014/164777, WO 95/10603, WO 95/26397, WO 96/23874, WO 96/23873, WO 97/41213, WO 99/19467, WO 00/60060, WO 00/29560, WO 99/23211 , WO99/46399, WO 00/60058, WO 00/60059, WO 99/42567, WO 01/14532, WO 02/092797, WO 01/66712, WO 01/88107, WO 01/96537, WO 02/10355, In WO 2006/002643, WO 2004/055178, and WO 98/13481.

Cellulase:

和

PREMIUM(诺维信公司)；

100、

200/220和

2000(美国丹尼斯克公司)；

(AB酶制剂公司(ABEnzymes))和KAC-500(B)(花王公司(Kao Corporation))。Cellulases can be added to the composition. Suitable cellulases include those of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Clostridium, Acremonium, for example, in e.g. Fungal fibers from Humicola insolens, Myceliophthorathermophila and Fusarium oxysporum disclosed in US Patent Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89/09259 Vegetase. Exemplary cellulases contemplated for use are those with color care benefits for textiles. Examples of such cellulases are those described, for example, in EP0495257, EP 0531372, WO 96/11262, WO 96/29397, and WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998; WO 98/12307; WO 95/24471; PCT/DK 98/00299; EP531315; Exemplary cellulases include those described in WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP0495257; and US Provisional Application Nos. 62/296,678 and 62/435340. Exemplary commercial cellulases include, but are not limited to

and

PREMIUM (Novozymes);

100,

200/220 and

2000 (Danisco, USA);

(AB Enzymes) and KAC-500(B) (Kao Corporation).

Mannanase:

Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as eg, WO2016007929; USPN 6566114, 6602842, and 6440991; and International Application Nos. PCT/US2016/060850 and PCT/US2016/ 060844. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as eg, WO 2016007929; USPN 6566114, 6602842, and 6440991; and International Application Nos. PCT/US 2016/060850 and PCT/US 2016 /060844.

Peroxidase/Oxidase:

Suitable peroxidases/oxidases contemplated for use in the compositions include those of plant, bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus (eg from C. cinereus), and variants thereof, eg WO 93/24618, WO 95/10602 , and those described in WO 98/15257. Commercially available peroxidases include, for example, GUARDZYME ^™ (Novo Nordisk and Novozymes).

The detergent composition may also comprise 2,6-beta-D-fructanohydrolase, which is effective for removing/cleaning biofilms present on household and/or industrial textiles/clothing.

One or more detergent enzymes may be included in the detergent composition by adding individual additives containing one or more enzymes, or by adding additives comprising a combination of all of these enzymes. Detergent additives, ie, individual additives or in combination, can be formulated, for example, as granules, liquids, slurries, and the like. Exemplary detergent additive formulations include, but are not limited to, granules (particularly dust-free granules), liquids (particularly stable liquids) or slurries.

Perhydrolase:

Perhydrolases include those described, for example, in WO 2005/056782, WO 2007/106293, WO 2008/063400, WO 2008/106214, and WO 2008/106215.

Nuclease:

Suitable nucleases include, but are not limited to, WO 2015/181287, WO 2015/155350, WO 2016/162556, WO 2017/162836, WO 2017/060475 (eg SEQ ID NO: 21), WO 2018/184816, WO 2018/177936 WO 2018/177938 , WO 2018/185280, and those described in WO 2018/206553.

Other nucleases that can be used in combination with the variant alpha-amylases of the invention include Nijland, R. et al. (2010) PLoS ONE [Public Library of Science: Comprehensive] 5-e15668 and Whitchurch, C.B. et al. (2002 ) those described in Science 295:1487.

4.6. Form of cleaning composition

The detergent composition may be in any convenient form, such as bars, tablets, powders, granules, pastes, or liquids. Liquid detergents can be aqueous and typically contain up to about 70% water and 0% to about 30% organic solvent. Compact detergent gels containing about 30% water or less are also contemplated. The variant alpha-amylases of the present invention are compatible with known forms and formulations of detergent compositions, and specific forms and formulations are described herein.

UltraPacks(汉高公司)、

Quantum(利洁时公司(Reckitt Benckiser))、CLOROX^TM 2Packs(高乐士公司(Clorox))、OxiClean Max Force Power Paks(杜威公司(Church&Dwight))、

Stain Release、

Pods、

ActionPacs、

Platimun、

和Pure essential(宝洁公司(Procter&Gamble))。单位剂量配制品和包装描述于例如US 20090209445 A1、US 20100081598 A1、US 7001878 B2、EP 1504994 B1、WO 2001085888 A2、WO 2003089562 A1、WO 2009098659 A1、WO 2009098660 A1、WO2009112992 A1、WO 2009124160 A1、WO 2009152031 A1、WO 2010059483 A1、WO2010088112 A1、WO 2010090915 A1、WO 2010135238 A1、WO 2011094687 A1、WO2011094690 A1、WO 2011127102 A1、WO 2011163428 A1、WO 2008000567 A1、WO2006045391 A1、WO 2006007911 A1、WO 2012027404 A1、EP 1740690 B1、WO 2012059336A1、US 6730646 B1、WO 2008087426 A1、WO 2010116139 A1、和WO 2012104613 A1中。A number of exemplary detergent formulations are described in WO 2013063460 to which an alpha-amylase of the present invention can be added (or in some cases the alpha-amylase is identified as a component of these formulations). These include commercially available unit dose detergent formulations/packages such as

UltraPacks (Henkel Corporation),

Quantum (Reckitt Benckiser), CLOROX ^™ 2Packs (Clorox), OxiClean Max Force Power Paks (Church & Dwight),

Stain Release,

Pods,

ActionPacs,

Platimun,

and Pure essential (Procter & Gamble).单位剂量配制品和包装描述于例如US 20090209445 A1、US 20100081598 A1、US 7001878 B2、EP 1504994 B1、WO 2001085888 A2、WO 2003089562 A1、WO 2009098659 A1、WO 2009098660 A1、WO2009112992 A1、WO 2009124160 A1、WO 2009152031 A1、WO 2010059483 A1、WO2010088112 A1、WO 2010090915 A1、WO 2010135238 A1、WO 2011094687 A1、WO2011094690 A1、WO 2011127102 A1、WO 2011163428 A1、WO 2008000567 A1、WO2006045391 A1、WO 2006007911 A1、WO 2012027404 A1、EP 1740690 B1 , WO 2012059336 A1, US 6730646 B1, WO 2008087426 A1, WO 2010116139 A1, and WO 2012104613 A1.

5. Carbohydrate Processing Using Variant Alpha-Amylases

Variant alpha-amylases can be used in a variety of industrial carbohydrate processing applications. For example, variant alpha-amylases can be used in starch conversion processes, particularly in saccharification processes that have undergone liquefaction. The desired end product can be any product that can be produced by enzymatic conversion of a starch substrate. For example, the desired product can be a glucose- and maltose-rich syrup that can be used in other processes (such as the production of HFCS), or can be converted into many other useful products, such as ascorbic acid intermediates (such as gluconic acid) salts; 2-keto-L-gulonic acid; 5-keto-gluconate; and 2,5-diketogluconate); 1,3-propanediol; aromatic amino acids (eg, tyrosine , phenylalanine, and tryptophan); organic acids (such as lactate, pyruvate, succinate, isocitrate, and oxaloacetate); amino acids (such as serine and glycine); antibiotics; Antimicrobials; Enzymes; Vitamins; and Hormones.

The starch conversion process can be a precursor to or concurrent with a fermentation process designed to produce alcohol for fuel or drinking (ie, drinkable alcohol). Those of ordinary skill in the art are aware of various fermentation conditions that can be used to produce these end products. Variant alpha-amylases can also be used in compositions and methods of food preparation. These various uses of variant alpha-amylases are described in more detail below.

5.1. Preparation of starch substrates

Methods for preparing starch substrates for use in the methods disclosed herein are known. Useful starch substrates can be obtained, for example, from tubers, roots, stems, legumes, grains or whole grains. More particularly, it can be obtained from corn, cob, wheat, barley, rye, triticale, milo, sago, millet, cassava, cassava, sorghum, rice, peas, kidney beans, bananas, or potatoes Granular starch. Particularly contemplated starch substrates are corn starch and wheat starch. Starch from grain can be ground or whole and includes corn solids such as kernel, bran and/or cobs. The starch can also be a highly refined raw starch or a raw material from a starch refining process.

5.2. Gelatinization and liquefaction of starch

Gelatinization typically occurs at the same time as or after the starch substrate is contacted with the alpha-amylase, although additional liquefaction-inducing enzymes may optionally be added. In some embodiments, the starch substrate prepared as described above is slurried with water. To optimize the stability and activity of the alpha-amylase, the pH of the slurry is typically adjusted to about pH 4.5-6.5, depending on the properties of the variant alpha-amylase used, and about 1 mM calcium (about 40 ppm) may also be added. of free calcium ions). The alpha-amylase remaining in the slurry after liquefaction can be inactivated via a number of methods, including lowering the pH in a subsequent reaction step or removing calcium from the slurry where the enzyme is calcium dependent. The starch plus alpha-amylase slurry can be pumped continuously through a jet cooker, which is steam heated to 105°C. The slurry was then cooled to room temperature.

5.3. Saccharification

Variant alpha-amylases can be used, optionally in the presence of another enzyme or enzymes, to saccharify liquefied starch into a syrup rich in low DP (eg, DP1 + DP2) sugars. The exact composition of the saccharified product depends on the combination of enzymes used and the type of granular starch being processed.

Liquefaction is usually carried out as a continuous process, while saccharification is usually carried out as a batch process. Saccharification is typically most effective at a temperature of about 60°C-65°C and a pH of about 4.0-4.5 (eg pH 4.3), it is necessary to cool the liquefied starch and adjust the pH of the liquefied starch. Saccharification is usually done in a stirred tank, which can take hours to fill or empty. Enzymes are typically added to the dry solids at a fixed rate (when the tank is being filled), or in a single dose (at the beginning of the filling phase). The saccharification reaction to make the syrup is typically carried out for about 24-72 hours, eg, 24-48 hours. For example, when the maximum or desired DE has been obtained, the reaction is stopped by heating to 85°C for 5 min. Further incubation will result in lower DE, eventually reaching about 90 DE, as the accumulated glucose repolymerizes to isomaltose and/or other reversal products via enzymatic reversal reactions and/or thermodynamic equilibrium methods.

5.4. Isomerization

IT(诺维信公司)；

IMGI、和

G993、

G993、

G993液体、和

IGI。异构化后，混合物典型地含有约40％-45％的果糖(例如42％的果糖)。Soluble starch hydrolysates produced by treatment with variant alpha-amylases can be converted into high fructose starch based syrups (HFSS) (eg high fructose corn syrup (HFCS)). This conversion can be achieved using glucose isomerase, in particular glucose isomerase immobilized on a solid support. The pH is increased to about 6.0 to about 8.0, eg, pH 7.5 (depending on the isomerase), and Ca2 ⁺ is removed by ion exchange. Suitable isomerases include

IT (Novozymes);

IMGI, and

G993,

G993,

G993 Liquid, and

IGI. After isomerization, the mixture typically contains about 40%-45% fructose (eg, 42% fructose).

5.5. Fermentation

Soluble starch hydrolysates, especially glucose-enriched syrups, can be fermented by contacting the starch hydrolysate with a fermenting organism, typically at a temperature of about 32°C, for example from 30°C to 35°C (for alcohol producing yeast) . The temperature and pH of the fermentation will depend on the fermenting organism. EOF products include metabolites such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone , sodium erythorbate, lysine and other amino acids, omega 3 fatty acids, butanol, isoprene, 1,3-propanediol and other biomaterials.

5.6. Combinations of variant alpha-amylases with other enzymes

300和OPTIDEX L-400(美国丹尼斯科公司)；AMIGASE^TM 和AMIGASE^TM PLUS(DSM)；

G900(酶生物系统公司(EnzymeBio-Systems))；和

G990ZR。Variant alpha-amylases may be combined with glucoamylases (EC 3.2.1.3). Exemplary glucoamylases are from Trichoderma, Aspergillus, Talaromyces, Clostridium, Fusarium, Thielavia, Thermomyces, Athelia, Humicola, Penicillium, Artomyces, Gloeophyllum, Pycnoporus, Steccherinum, Trametes, etc. Suitable commercial glucoamylases include AMG 200L; AMG 300L; SAN ^™ SUPER and AMG ^™ E (Novozymes);

300 and OPTIDEX L-400 (Danisco, USA); AMIGASE ^TM and AMIGASE ^TM PLUS (DSM);

G900 (Enzyme Bio-Systems); and

G990ZR.

Other suitable enzymes that can be used with variant alpha-amylases include phytases, proteases, pullulanases, beta-amylases, isoamylases, alpha-glucosidases, cellulases, xylanases , other hemicellulases, beta-glucosidases, transferases, pectinases, lipases, cutinases, esterases, oxidoreductases, various alpha-amylases, or combinations thereof.

Compositions comprising the alpha-amylases of the present invention may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further comprise any of the additional enzymes listed herein or Various, as well as buffers, salts, preservatives, water, co-solvents, surfactants, etc.

6. Textile desizing compositions and uses

Also contemplated are compositions and methods for treating fabrics (eg, desizing textiles) using amylases. Fabric treatment methods are well known in the art (see, eg, US Pat. No. 6,077,316). For example, the hand and appearance of the fabric can be improved by a method comprising contacting the fabric with alpha-amylase in solution. The fabric can be treated with the solution under pressure.

The alpha-amylase may be applied during or after weaving of the textile or during the desizing stage or in one or more additional fabric processing steps. During the weaving of textiles, the threads are exposed to considerable mechanical strain. Before weaving on mechanical looms, warp yarns are usually coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. Alpha-amylases can be applied during or after weaving to remove these sizing starches or starch derivatives. After weaving, alpha-amylase can be used to remove the size coating prior to further processing the fabric to ensure a uniform and washable result.

Alpha-amylases can be used alone or in combination with other desizing chemicals and/or desizing enzymes to desize fabrics, including cotton-containing fabrics, as detergent additives (eg, in aqueous compositions). Alpha-amylases can also be used in compositions and methods for producing a stone-milled look on indigo dyed denim fabrics and garments. For garment production, fabrics can be cut and sewn into garments or garments, which are subsequently finished. In particular, for the production of denim, different enzymatic finishing methods have been developed. Finishing of denim garments typically begins with an enzymatic desizing step, in which the garment is subjected to amylolytic enzymes to provide softness to the fabric, making the cotton more susceptible to subsequent enzymatic finishing steps. Alpha-amylases can be used in the following methods: finishing denim garments (eg "bioscrub process"), enzymatic desizing and providing softness to fabrics and/or finishing methods.

7. Compositions and methods for baking and food preparation

The compositions and methods of the present invention also relate to food compositions including, but not limited to, food, animal feed and/or food/feed additives (including variant alpha-amylases), and methods for preparing such food compositions (The method comprises admixing a variant alpha-amylase with one or more food ingredients), or use thereof. Furthermore, the compositions and methods of the present invention relate to baking compositions (including but not limited to baked goods flours, doughs, baking additives and/or baked products).

9. Brewing composition

The variant alpha-amylases of the present invention may be a component of a brewing composition used in a brewing process (ie making a fermented malt beverage). Non-fermentable carbohydrates form most of the dissolved solids in the final beer. This residue persists because maltoamylase is unable to hydrolyze the alpha-1,6-bonds of starch. Alpha-amylase, optionally in combination with glucoamylase and optionally pullulanase and/or isoamylase, helps convert starch to dextrins and fermentable sugars, reducing residual non- Fermentable carbohydrates.

All references cited herein are incorporated by reference in their entirety for all purposes. In order to further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative and not restrictive.

example

Example 1. AA2560 variant

Protein expression, purification and quantification:

The AA2560 combinatorial variants in the ΔR181 and ΔG182 (ie ΔRG) backgrounds were made into synthetic genes and introduced into suitable Bacillus licheniformis cells using standard procedures. All mutations were confirmed by DNA sequencing. Cells were grown for 72 hours in medium suitable for protein expression and secretion in B. licheniformis hosts. Secreted proteins are harvested by centrifugation. Purification was achieved by hydrophobic interaction chromatography with Phenyl Sepharose 6 Fast Flow Resin (GE Healthcare). The purified protein was stabilized in standard formulation buffer (pH 8) containing HEPES (as buffer), calcium chloride and propylene glycol. Protein concentration was determined by using amino acid analysis, high performance liquid chromatography (HPLC) and absorbance at 280 nm simultaneously.

Enzyme performance assay:

Alpha-amylase activity was determined by removing dyed starch stains from white melamine tiles in a detergent background. Mixed corn/rice colored starch bricks and mixed corn/rice starch bricks with food colorants (Cat. Nos. DM277 and DM71), respectively, purchased from the Center for Testmaterials, were used to determine the cleaning activity of alpha-amylases. Bricks were mounted on 96-well plates (containing amylase solution diluted to the working range in aqueous buffer) and added to a pre-made detergent solution of WFKB detergent (WFK Testgewebe, Brüggergen, Germany), Make the total volume 300 μL. The pre-imaged melamine tiles containing the colored starch stains were then mounted on the top of the 96-well plate so that the stirring assembly allowed the enzymatic detergent to be splashed onto the surface of the starch stains. Washing reactions were performed at 50°C for 15 minutes with shaking at 250 rpm. After the wash reaction, the melamine tiles were then briefly rinsed under water, dried and reimaged. Alpha-amylase activity was calculated as the difference between the RGB (color) values of the images before and after washing. The whiter the image after washing, the higher the enzyme activity. The Performance Index (PI) is calculated as:

Performance metrics of the combined variant relative to the ΔRG variant:

The cleaning performance of the variants in terms of performance index relative to the wild type variant is listed in Table 3. DM277 was named Stain 1 and DM71 was named Stain 2.

Table 3. Variant performance for two different stains

mutation PI-1 PI-2 ΔR181-ΔG182 -1- -1- T40N-S91R-Q169H-ΔR181-ΔG182-T183M-H281N 1.18 1.60 Q172R-ΔR181-ΔG182-E190P-S288D 1.09 1.36 Q172R-ΔR181-ΔG182-S244E-S288D-S474R 1.15 1.82 S91R-Q172R-ΔR181-ΔG182-E190P-I324M 1.26 1.95 T40N-S91R-ΔR181-ΔG182-E190P-F263Y 1.20 1.99 T40N-S91R-ΔR181-ΔG182-S244E-Y364L 1.20 2.19 S91R-Q172R-ΔR181-ΔG182-E190P-I324R 1.26 1.84 S91R-W116R-Q172R-ΔR181-ΔG182-S244E-H281S-S288D 1.29 2.19 T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S 1.24 1.88 T40N-S91R-Q172R-ΔR181-ΔG182-S244Q-F263Y-H281S 1.19 1.94 S91R-Q172R-ΔR181-ΔG182-E190P-I324N 0.96 1.44 T40D-S91R-Q172R-ΔR181-ΔG182-E190P-H281S-I324R 1.04 0.99 ΔR181-ΔG182-Y364L 2.90 2.23

Plus 12L(诺维信公司)。All variants in Table 2 performed equal to or better than one or more of the stains and wash conditions tested

Plus 12L (Novozymes).

Example 2. Additional AA2560, AA707 and AAI10 variants

To demonstrate the beneficial effect of the combinatorial mutations described in Example 1 in other parental molecules, additional AA2560, AA707 and AAI10 variants were prepared and tested as described. These molecules additionally included deletions at positions 181 and 182, 183 and 184, or no deletions. All mutations were confirmed by DNA sequencing.

The performance of the variants is measured relative to the closest parent, ie combinatorial variants in "non-deletion" molecules are measured relative to the respective wild-type molecule. Combinatorial variants in "double deletion" molecules are measured relative to the respective double deletion molecule. The results are shown in the table of FIG. 5 .

The results show that, with the exception of AA707, mutations between different parental molecules are highly transferable. An explanation for the different behavior of AA707 for mutations at the Ser91 position is presented in the Detailed Description section herein.

Claims (28)

1. A recombinant, non-naturally occurring parent variant alpha-amylase molecule comprising a mutation at position 91 numbered with reference to SEQ ID NO: 1 and an amino acid residue at the bottom of the alpha-amylase TIM barrel structure. Mutations, the bottom amino acid residues are defined as residues 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325 , 362, 363, and 364, wherein the wild-type amino acid residue present at position 28 of the parent molecule is positively charged. 2. The variant alpha-amylase of claim 1, wherein the mutation at position 91 replaces the naturally occurring residue with a positively charged residue. 3. The variant alpha-amylase of claim 1 or 2, wherein the mutation at position 91 replaces the naturally occurring residue with an arginino group (ie, X91R). 4. The variant alpha-amylase of any one of claims 1-3, wherein at least one mutation at the bottom of the alpha-amylase TIM barrel structure is selected from the group consisting of: X40N, X40D, X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L, and X364M. 5. The variant alpha-amylase of any one of claims 1-4, wherein at least one mutation at the bottom of the alpha-amylase TIM barrel structure is selected from the group consisting of: T40N, T40D, Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L, and Y364M. 6. A recombinant, non-naturally occurring variant alpha-amylase comprising arginine at position 91 and at least one of the following features not present in the naturally occurring alpha-amylase: position 40 N or D, F or L at position 100, Y at position 263, D, K or Q at position 288, R, N or M at position 324, or L or M at position 364. 7. The variant alpha-amylase of any one of claims 1-6, further comprising mutations at residues in a loop comprising surface exposed residues numbered with reference to SEQ ID NO: 1 Bases 167, 169, 171, 172, and 176. 8. The variant alpha-amylase of claim 7, wherein at least one mutation in the loop is selected from the group consisting of X167F, X169H, X171Y, X172R, X172N, and X176S. 9. The variant alpha-amylase of claim 8, wherein at least one mutation in the loop is selected from the group consisting of: W167F, Q169H, R171Y, Q172R, Q172N, and R176S. 10. The variant alpha-amylase of any one of claims 1-6, further comprising F at position 167, H at position 169, H at position 171 numbered with reference to SEQ ID NO: 1 Y, R or N at position 172, or S at position 176. 11. A recombinant, non-naturally occurring variant alpha-amylase comprising a mutation at position 172 and a mutation at position 288 numbered with reference to SEQ ID NO:1. 12. A recombinant, non-naturally occurring variant alpha-amylase comprising arginine or asparagine at position 172 and aspartic acid at position 288 as numbered with reference to SEQ ID NO:1. 13. The variant alpha-amylase of any one of claims 1-12, further comprising mutations at positions 116 and/or 281 numbered with reference to SEQ ID NO:1. 14. The variant alpha-amylase of any one of claims 1-12, further comprising an arginine at position 116 or a serine at position 281 numbered with reference to SEQ ID NO:1. 15. The variant alpha-amylase of any one of claims 1-14, further comprising mutations at positions 190 and/or 244 numbered with reference to SEQ ID NO:1. 16. The variant alpha-amylase of any one of claims 1-14 having a proline at position 190 and/or alanine at position 244 numbered with reference to SEQ ID NO: 1 , glutamate or glutamine. 17. The variant alpha-amylase of any one of claims 1-16, further comprising using SEQ ID NO: 1 corresponding to at least two residues of R181, G182, T183, and G184 missing. 18. The variant alpha-amylase of any one of claims 1-16, further comprising a pairwise deletion of residues corresponding to R181 and G182 or residues T183 and G184. 19. A recombinant, non-naturally occurring variant alpha-amylase comprising Substitutions of (i) numbered using SEQ ID NO: 1 are selected from the group consisting of: (a) X40N-X91R-X169H-X183M-X281N, (b) X172R-X190P-X288D, (c) X172R-X244E-X288D-X474R, (d) X91R-X172R-X190P-X324M, (e) X40N-X91R-X190P-X263Y, (f) X40N-X91R-X244E-X364L, (g) X91R-X172R-X190P-X324R, (h) X91R-X116R-X172R-X244E-X281S-X288D, (i) X40N-X91R-X100F-X116R-X172N-X244Q-X281S, (j) X40N-X91R-X172R-X244Q-X263Y-X281S, (k) X91R-X172R-X190P-X324N, (l) X40D-X91R-X172R-X190P-X281S-X324R, and (m)X364L; and (ii) pairwise deletion of residues selected from the group consisting of residues corresponding to: 181 and 182, and 183 and 184. 20. The recombinant, non-naturally occurring variant alpha-amylase of claim 19, comprising Substitutions of (i) numbered using SEQ ID NO: 1 are selected from the group consisting of: (a) T40N-S91R-Q169H-T183M-H281N, (b) Q172R-E190P-S288D, (c) Q172R-S244E-S288D-S474R, (d) S91R-Q172R-E190P-I324M, (e) T40N-S91R-E190P-F263Y, (f) T40N-S91R-S244E-Y364L, (g) S91R-Q172R-E190P-I324R, (h) S91R-W116R-Q172R-S244E-H281S-S288D, (i) T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S, (j) T40N-S91R-Q172R-S244Q-F263Y-H281S, (k)S91R-Q172R-E190P-I324N, (l) T40D-S91R-Q172R-E190P-H281S-I324R, and (m) Y364L; and (ii) pairwise deletion of residues selected from the group consisting of: R181 and G182, and T183 and G184. 21. A recombinant, non-naturally occurring variant alpha-amylase comprising three or more of the following features: (a) D or N at position 40 and/or R at position 91, and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and/or L at position 364, optionally with (c) position 169 H at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R at position 474, and/or Combinations of R at position 116, (d) optionally with (e) paired deletions at positions 181 and 182 or 183 and 184, in all cases using SEQ ID NO: 1 for numbering. 22. The variant alpha-amylase of any one of claims 1-21, which corresponds to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 have at least 70%, at least 80%, at least 90% or at least 95% amino acid sequence identity. 23. A detergent composition comprising the variant alpha-amylase of any one of claims 1-22. 24. The detergent composition of claim 23, further comprising a variant subtilisin from Bacillus gibsonii, the variant subtilisin having amino acid substitutions X39E, X99R, X126A, X127E, and X128G, and further comprise one or more additional substitutions numbered with reference to SEQ ID NO: 5 selected from the group consisting of: N74D-M211L-N253P, R179Q-M211L-N253P, N74D-N253P, N85R-G160Q- R179Q-M211L-N12S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N253P, R179Q-M211L, G160Q-R179Q-M253P, G160Q-N253P, N74D-M211L G160Q-R179Q-M211L-N212S, N74D-R179Q-M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N74D-Q200L-M211L, N74D-G160Q-N212S-N253P-M211-N74D G160Q-R179Q, G160Q-R179Q-N212S, N74D-G160Q-N253P, N74D-G160Q-R179Q-M211L-N212S-N253P, N74D-N085R-G160Q-R179Q-M211L, N74D-G160-N212SL N085R-N116R-Q200L-Q256E, N74D-G160Q-R179Q-N212S-N253P, N74D-G160Q-M211L-N212S, N74D-G160Q, N74D-G160Q-R179Q-Q211L-N253P, N74D-4179Q N212S, N74D-G160Q-M211L, N74D-G160Q-R179Q-N253P, N74D, N74D-G160Q-R179Q-M211L-N212S, N74D-N085R-M211L-N212S, N74D-G160Q-R179Q-N212S, M211L, N74D-M211L-Q256E, N74D-G160Q-R179Q, R179Q-M211L-N212S-N2 53P, R179Q-M211L-N212S, N74D-N085R-R179Q-M211L-N212S, N74D-M211L-N212S, N74D-R179Q-M211L-N212S, N74D-M211L-N242D, N74D-Q200L-M211L-N212S M211L-N242D-Q256E, N74D-Q200L, N74D-M211N-N212Q, N74D-M211N-N212Q-Q256E, N74D-M211N-Q256E, N74D-M211Q, N74D-M211Q-N212Q, N74Q-M211Q-N212 M211Q-Q256E, N74D-N198A-M211Q, N74D-N198A-M211Q-N212Q, N74D-N198A-M211Q-Q256E, N74D-N198G-M211Q, N74D-N198G-M211Q-N212Q, N74Q256E, N74Q-N198G-DM211Q N198K-M211Q-N212Q, N74D-N198L-M211Q-N212Q, N74D-N198Q-M211Q-N212Q, N74D-N198R-M211Q-N212Q, N74D-N198T-M211Q-N212Q, N74D-N198V-M4111Q Q256E, N74D-Q256E, N74D-R207Q, N74D-R207Q-M211N, N74D-R207Q-M211N-N212Q, N74D-R207Q-M211N-N212Q-Q256E, N74D-R207Q-M211N-Q256E, N74D-R207 R207Q-M211Q-N212Q, N74D-R207Q-M211Q-N212Q-Q256E, N74D-R207Q-N212Q, N74D-R207Q-N212Q-Q256E, N74D-R207Q-Q256E, N74D-N198S-M211Q, and N74D, Has at least 90% amino acid sequence identity with SEQ ID NO:6. 25. A method for converting starch to oligosaccharides, the method comprising contacting starch with an effective amount of the variant alpha-amylase of any one of claims 1-22. 26. A method for removing starch stains or soils from a surface, the method comprising contacting the surface with an effective amount of the variant alpha-amylase of any one of claims 1-22, and The starch stain is removed from the surface by allowing the polypeptide to hydrolyze the starch components present in the starch stain to produce smaller starch-derived molecules that dissolve in the aqueous composition. 27. A nucleic acid encoding the variant alpha-amylase of any one of claims 1-22. 28. A host cell comprising the nucleic acid of claim 27.

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